Identification and Characterization of Germline Mutations in Indian Women with Cancer: A Tertiary Center Next-Generation Sequencing Study

Abstract

Introduction

Hereditary cancer syndromes account for 5 to 10% of all malignancies, with BRCA1/2 and mismatch repair (MMR) genes playing crucial roles in breast, ovarian, and endometrial cancers. However, the prevalence and spectrum of germline mutations in Indian women remain inadequately characterized.

Objective

The aim of the study was to determine the proportion of patients harboring pathogenic or likely pathogenic (P/LP) variants and compare the mutation frequencies across cancer subtypes to help provide evidence for genetic counseling, cascade screening, and development of population-specific variant annotation resources to enhance precision oncology in India.

Materials and Methods

We prospectively enrolled 244 histopathologically confirmed cancer patients (193 women and 51 men) meeting National Comprehensive Cancer Network criteria for germline testing (August 2022–June 2025). Genomic DNA from peripheral blood underwent targeted enrichment using the GALEAS HereditaryPlus 146-gene panel and next-generation sequencing on an Illumina NextSeq 2000 platform (mean depth ∼500×). Variants were bioinformatically processed, aligned to the GRCh38 reference genome, and classified according to the guidelines of the American College of Medical Genetics and Genomics.

Results

P/LP variants were identified in 55/193 women (28.5%) and 6/51 men (11.8%). Among women, the highest mutation rates were observed in endometrial (30.0%), ovarian (28.6%), and breast (28.0%) cancers. BRCA1 (10.9%) and BRCA2 (6.7%) mutations predominated, followed by MMR genes (MLH1, MSH2, MSH6; cumulative 4.7%). Triple-negative breast cancer exhibited the highest mutation burden (35.1%, p < 0.01). Variants of uncertain significance were detected in 8.8% of women (17/193), highlighting the need for population-specific annotation resources.

Conclusion

More than one-quarter of Indian women with major gynecological malignancies harbor actionable germline variants. Our findings underscore the importance of routine multigene panel testing, systematic cascade screening in at-risk relatives, and the development of India-specific variant databases to facilitate precision oncology.

Keywords

germline mutation - hereditary cancer - BRCA1/2 - mismatch repair genes - next-generation sequencing - Indian population

Introduction

Cancer represents a leading cause of morbidity and mortality worldwide, with distinctive epidemiological patterns across geographical regions and populations. According to GLOBOCAN 2022, approximately 19.98 million new cancer cases and 9.74 million cancer-related deaths were reported globally.[1] [2] Cancer burden exhibits substantial geographic and gender-specific variation, with developed regions such as the United States, China, and Europe generally reporting higher cancer incidence rates in males than females.

Notably, India demonstrates a distinctive epidemiological profile characterized by a higher cancer incidence in females (55.5%) compared with males (45.5%), with an overall burden of 7.1%.[3] The National Cancer Registry Programme Report (2022) estimated 1.46 million incident cancer cases in India, with a higher crude rate in females (105.4 per 100,000) than males (95.6 per 100,000). This contrasts with global patterns, where male cases (9.57 million) slightly exceed female cases (9.18 million).[2]

Female-specific cancers in India, notably breast, cervical, and ovarian, account for a disproportionately high share of the cancer burden. This unique distribution is influenced by multiple factors, including indoor air pollution from biomass fuel combustion, reproductive patterns (early marriage, high parity, limited contraceptive use), low human papillomavirus vaccination coverage, and limited access to screening programs. These factors collectively shape the distinct cancer epidemiology observed among Indian women compared with Western populations.[4] [5]

While environmental and lifestyle factors substantially influence cancer risk, hereditary genetic factors significantly contribute to 5 to 10% of all malignancies.[6] Germline mutations in high-penetrance genes such as BRCA1 and BRCA2 are well-established drivers of hereditary breast and ovarian cancer (HBOC) syndrome, conferring lifetime breast cancer risks up to 72% in mutation carriers.[7] [8] Additional hereditary cancer syndromes arise from mutations in genes including TP53 (Li–Fraumeni's syndrome), PALB2, CHEK2, and mismatch repair (MMR) genes such as MLH1, MSH2, and MSH6 (Lynch syndrome).[9]

Despite growing evidence globally, hereditary cancer studies in Indian populations remain limited. Existing reports indicate variable BRCA mutation frequencies ranging from 15 to 30% in high-risk families, with considerable regional heterogeneity. The spectrum of non-BRCA pathogenic (P) variants and the prevalence of variants of uncertain significance (VUS) in the Indian context remain inadequately characterized, complicating genetic counseling and clinical management.[10]

Comprehensive germline mutation profiling in Indian cancer patients is essential for multiple reasons: (1) it supports cascade testing and genetic counseling, promoting early diagnosis in families; (2) population-specific mutation data improve variant classification and clinical guideline development; (3) identification of P variants enables targeted therapeutic approaches, including PARP inhibitors for BRCA-mutated tumors and immunotherapy for MMR-deficient cancers; and (4) it informs public health strategies for cancer prevention and early detection.[11]

To address these knowledge gaps, we conducted a comprehensive next-generation sequencing (NGS) analysis using a 146-gene panel at a tertiary care hospital, where highly specialized treatment facilities and settings enabled comprehensive genetic testing and detection of hereditary mutations compared with primary or secondary care centers. This study represents one of the largest multigene germline analyses in Indian women with cancer (i.e., breast, ovarian, endometrial, and related cancers), carried out in accordance with Indian Society of Medical and Paediatric Oncology guidelines, aimed at characterizing the prevalence, spectrum, and clinical implications of hereditary cancer predisposition.[12]

Materials and Methods

Study Design

This prospective observational study was conducted between August 2022 and June 2025 at the School of Medicine, Amrita Vishwa Vidyapeetham, Faridabad, India. Histopathologically confirmed cancer patients who met National Comprehensive Cancer Network (NCCN) criteria were enrolled for this study.

Patient Selection and Eligibility Criteria

Participants were selected based on the NCCN guidelines for germline genetic testing. Inclusion criteria comprised: (1) histopathologically confirmed malignancy and (2) fulfillment of NCCN criteria for hereditary cancer risk assessment. Participants excluded from this study included patients with insufficient clinical or family history data for hereditary risk assessment, individuals who declined genetic counseling, duplicate samples from the same individual, and inadequate or poor-quality DNA samples.

A total of 244 cancer patients were enrolled, comprising 193 females and 51 males (included for comparison but not the primary focus). The female cohort included 75 patients with breast cancer, 63 with ovarian cancer, 30 with endometrial cancer, and 25 with other malignancies. Details of number of patients and cancer type are mentioned in [Table 1].

Table 1 Population statistics and results of germline testing, stratified by cancer type, for patients included within this study (n = 244)

Group	Cancer type	N	Age (range)	Path/LP, N (%)	VUS, N (%)	Negative, N (%)
Females	Breast cancer	75	45.2 (23–75)	21 (28.0%)	4 (5.3%)	50 (66.7%)
	Ovarian cancer	63	53.65 (22–76)	18 (28.6%)	6 (9.5%)	39 (61.9%)
	Endometrial cancer	30	57.86 (31–73)	9 (30.0%)	3 (10.0%)	18 (60.0%)
	Other cancers	25	46.48 (18–76)	7 (28.0%)	4 (16.0%)	14 (56.0%)
Total		193	50.80 (18–76)	55 (28.5%)	17 (8.8%)	121 (62.7%)
Males	Prostate cancer	26	63.8 (33-85)	0 (0.0%)	4 (15.4%)	22 (84.6%)
	Colorectal cancer	8	48.0 (12-75)	2 (25.0%)	0 (0.0%)	6 (75.0%)
	Renal cancer	6	38.17 (26–48)	2 (33.3%)	0 (0.0%)	4 (66.7%)
	Lung cancer	3	62 (57–69)	0 (0.0%)	2 (66.7%)	1 (33.3%)
	Other cancers	8	41.5 (10–66)	2 (25.0%)	0 (0.0%)	6 (75.0%)
Total		51	54.0 (10–85)	6 (11.8%)	6 (11.8%)	39 (76.5%)

Note: The odds of finding male patients harboring detectable, actionable mutations (pathogenic or likely pathogenic) were significantly lower than female patients (p < 0.05), highlighting the importance of germline testing for female populations, particularly.

Sample Collection and DNA Extraction

Peripheral blood (3 mL) was collected in EDTA vacutainers from each participant. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer's protocol. DNA concentration and quality were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, United States), Qubit 4 Fluorometer (Thermo Fisher Scientific), and Agilent 4200 TapeStation for fragment size distribution. Only samples with an A260/280 ratio between 1.8 and 2.0 and adequate DNA yield (≥ 50 ng) were processed for sequencing.

Target Enrichment and Library Preparation

Germline mutation profiling was performed using the GALEAS HereditaryPlus Kit (Nonacus, UK), which targets 146 genes associated with hereditary cancer syndromes. Approximately 50 ng of genomic DNA per sample was used for library preparation following the manufacturer's protocol (Nonacus, GALEAS HereditaryPlus Protocol Guide v1.1, cat: NGS_GAL_HCP_FR_96). The protocol included DNA fragmentation, end repair, adapter ligation, and polymerase chain reaction amplification. The fragmented DNA was hybridized with biotin-labeled probes targeting the regions of interest, followed by enrichment using streptavidin-coated Dynabeads M-270. The captured DNA was amplified using Illumina-specific primers and purified with Target Pure NGS clean-up beads to remove nonspecific products and primer dimers.

Next-Generation Sequencing

Paired-end sequencing (2 × 150 bp) was performed on an Illumina NextSeq 2000 system. Libraries were sequenced to achieve a minimum mean coverage depth of ×500, with > 95% of target regions covered at ≥ ×250. Sequencing quality metrics, including Q30 scores, cluster density, and total reads, were monitored to ensure optimal data generation. The sequencing libraries were prepared following the manufacturer's guidelines for optimal data generation. Samples were demultiplexed based on assigned indexes, and FASTQ files were saved for secondary analysis.

Bioinformatics Analysis and Variant Classification

Secondary analysis of the obtained FASTQ files was performed with NONACUS GALEAS, which is a web-based portal. The software performs quality control (QC), alignment, and generates Variant Call Format files for single-nucleotide variants (SNVs), indels, and copy number variants (CNVs). QC and adapter trimming were performed using Trimmomatic (v0.40). High-quality reads were aligned to the human reference genome (GRCh38/hg38) using BWA-MEM (v0.7.17), followed by postalignment processing with GATK (v4.5.0).

Variant calling was performed using GATK Haplotype Caller for SNVs and small insertions/deletions (indels). CNVs were detected using ExomeDepth and validated by multiplex ligation-dependent probe amplification where applicable. Variants were filtered based on quality metrics: minimum read depth of ×500 and variant allele frequency ≥ 0.3.

Annotation was performed using VarSome Clinical (v12.5), incorporating data from ClinVar (June 2025), gnomAD v4.0 (South Asian subset), and in silico prediction tools (CADD, REVEL, SpliceAI). Variants were classified according to the American College of Medical Genetics and Genomics guidelines into five categories: P, likely pathogenic (LP), VUS, likely benign, and benign (B).

Statistical Analysis

Descriptive statistics were used to summarize patient demographics and mutation frequencies across different cancer types. Proportions of P, LP, and VUS variants were calculated for each gene and cancer subtype. Associations between mutation status and clinical variables (age, receptor status, microsatellite status, etc.) were analyzed using chi-square or Fisher's exact tests as appropriate. Continuous variables were compared using the Kruskal–Wallis' test or the Mann–Whitney's U test. A p-value of < 0.05 was considered statistically significant. All statistical analyses were conducted using R software (version 4.5.0).

Ethical Approval

The study has been conducted in accordance with the Helsinki Declaration. The study protocol was approved by the Institutional Ethics Committee (IEC approval number: ECASM-AIMS-2023-193).

Patient Counseling and Ethical Approval

Pre- and posttest genetic counseling was provided to all participants by trained clinicians or certified genetic counselors. Each participant received information about the purpose, scope, benefits, and limitations of genetic testing, followed by written informed consent in accordance with institutional ethics policy.

Results

Patient Demographics and Cancer Distribution

Among the 193 female patients included in the final analysis, the median age was 50.8 years (range: 18–76 years). Breast cancer (n = 75, 38.86%), ovarian cancer (n = 63, 32.64%), and endometrial cancer (n = 30, 15.54%) were the predominant malignancies, collectively accounting for 87.04% of cases ([Fig. 1]). Other cancers (n=25, 12.96%) included cancers of colorectal (n=3), lung (n=3), bone (n=4), brain (n=1), pancreas (n=2), liver (n=2), Hodgkin's lymphoma (n=1), rectum (n=1), stomach (n=1), unknown primary (n=2), thyroid (n=1), acute lymphoid leukemia (n=1), and renal malignancies (n=3). The distribution of P/LP variants across different cancer types is presented in [Table 1].

Fig. 1 (A) Boxplot showing average age ranges in each cancer type in the female cohort, and individual cases are shown as jitter over the boxplot. (B) Percentage distribution of each gene in overall cases included in the study for pathogenic (left) and (C) VUS (right). BRCA1 has the highest percentage (10.88%) of pathogenic variants, followed by BRCA2 (6.74%). VUS, variants of uncertain significance

Spectrum and Distribution of Germline Mutations

Among the 55 female patients (28.5%) harboring detectable P/LP germline alterations, a total of 56 distinct variants were identified, with one patient carrying two P/LP variants. These comprised 44 P and 12 LP variants distributed across multiple genes (variant-level details for each mutation are provided in the [Supplementary Material]).

Furthermore, 17 patients with detectable mutations were classified as VUS. No discernible patterns or associations were seen in mutations in patients harboring multiple mutations.

The most frequently mutated genes were BRCA1 (10.9% of the total cohort, n = 21) and BRCA2 (6.7%, n = 13), followed by MMR genes including MLH1, MSH2, and MSH6 (each 1.6%, cumulative 4.7%). Other high-penetrance genes with identified mutations included TP53 (1.0%), PTEN (0.5%), and PALB2 (0.5%). Moderate- to low-penetrance genes with P/LP variants included NBN, POLE, FANCA, TSC2, RB1, and VHL, each observed in a single patient (0.5%). These results are summarized in [Table 2].

Table 2 Genes with pathogenic, likely pathogenic, and VUS detected within the scope of this study across cancer types

Cancer type	N (%)	Genes with pathogenic variants	Genes with VUS
Breast	75 (38.86%)	BRCA1, BRCA2, PTEN, MSH2, FANCA, PALB2, NBN	RAD50, POLD1, CHEK2, BRCA1, BRCA2, MSH6
Ovarian	63 (32.64%)	BRCA1, BRCA2, TP53, TSC2	BRCA2, RAD51D, BRCA1, RAD52, BAP1, MSH6, RAD54L, BARD1
Endometrial	30 (15.54%)	MLH1, MSH6, MSH2, BRCA1	MUTYH, APC, PMS2, RAD51D, CHEK2
Others	25 (12.95%)	POLE, NBN, BRCA2, RB1, VHL, TSC2	BRCA1, BRCA2, POLD1, ATM, FH
Across all subtypes	193	BRCA1, BRCA2, MLH1, MSH2, MSH6, NBN, POLE, PTEN, RB1, TP53, VHL, FANCA, TSC2, PALB2	BRCA2, BRCA1, RAD51D, CHEK2, MSH6, POLD1, RAD50, APC, ATM, BARD1, BAP1, MUTYH, PMS2, RAD52, RAD54L, FH

Abbreviation: VUS, variants of uncertain significance.

The spectrum of P variants in BRCA1 included 10 frameshift, 7 nonsense, and 4 splice-site mutations (see [Fig. 2]). BRCA2 variants comprised eight frameshift, three missense with proven functional impact, and two splice-site mutations (see [Fig. 2] ). In BRCA1, the highest frequency of P variants occurred in exon 11, which represents the largest coding region, with additional enrichment within the N-terminal RING finger domain (exons 2–7) and the C-terminal BRCT repeats (exons 16–24), both critical for tumor suppressor function. In BRCA2, mutations clustered predominantly within the BRC repeat region (exons 11–14), which mediates RAD51 binding, and the C-terminal DNA-binding domain (exons 17–27), encompassing the helical and OB folds essential for homologous recombination. This distribution highlights exon 11 as a mutational hotspot in both BRCA1 and BRCA2, while emphasizing functional domains as key regions of vulnerability.

Fig. 2 (A) BRCA1 variants (n = 11) mapped across the 1,863-amino acid protein, showing canonical domains: RING domain (amino acids 24–64), coiled-coil region (1,364–1,437), and BRCT domains 1 and 2 (1,646–1,736 and 1,760–1,855, respectively). (B) BRCA2 variants (n = 16) mapped across the 3,418-amino acid protein, displaying functional regions including the transactivation core (1–250), BRC repeats (1,009–2,083), DNA-binding domain (2,479–3,186), and RAD51/NLS-binding region (3,265–3,330).

Cancer-Specific Mutation Profiles

Breast Cancer

In the breast cancer cohort (n = 75, 38.86%), invasive carcinoma was the predominant histological subtype (90.5%), and 86% of patients presented with advanced disease (stage III or IV). Based on immunohistochemistry (IHC), the cohort was classified into molecular subtypes: Luminal A (ER/PR + , HER2 − ), Luminal B (ER/PR + , HER2 + ), HER2-enriched (ER/PR − , HER2 + ), and triple-negative breast cancer (TNBC).

P/LP germline variants were identified in 21 patients (28.0%), with 70% (n = 14) diagnosed before the age of 45 years. BRCA1 mutations were most common (n = 9, 12.2%), followed by BRCA2 (n = 6, 8.1%). Single cases (1.4% each) harbored mutations in PTEN, MSH2, FANCA, PALB2, and NBN.

The triple-negative subtype exhibited the highest prevalence of P germline mutations, with 35.1% of TNBC cases (13/37) carrying P/LP variants compared with 21% (8/38) in non-TNBC cases. As confirmed in other studies, BRCA1 mutations were significantly enriched in early-onset disease (< 45 years) compared with later-onset cases (relative risk 2.1, 95% confidence interval 1.1–4.0).

Ovarian Cancer

Among 63 (32.64%) women with ovarian carcinoma, high-grade serous histology was predominant (87.3%), and 95% were diagnosed at International Federation of Gynaecology and Obstetrics (FIGO) stage III or IV. P/LP germline variants were observed in 18 patients (28.6%). The most common were BRCA1 (17.5%, n = 11) and BRCA2 (9.5%, n = 6), followed by TP53 and TSC2 (1.6% each).

VUS were observed in BRCA2 (6.4%, n = 4), and single cases (1.6% each) harbored VUS in RAD51D, RAD52, BAP1, MSH6, RAD54L, and BARD1.

Endometrial Cancer

Among 30 (15.54%) women with endometrial cancer, IHC for MMR proteins was available for all patients; 27 patients showed loss of one or more of the MMR genes (details in [Supplementary Material], available in online version only). Among tumors with demonstrable MMR loss, 29.6% (8/27) showed loss concordant with the mutated gene, supporting functional impact. All 30 patients underwent comprehensive hereditary germline testing using a 146-gene panel as described in the Materials and Methods section. P/LP variants were identified in MMR genes, MLH1 (n = 3), MSH6 (n = 3), and MSH2 (n = 2). In one additional patient, P BRCA1 variant was identified with retained MMR on IHC. Most patients with P variants (8/9, 88.9%) were diagnosed after the age of 45 years, contrasting with the younger age distribution observed in breast cancer, where 70% of mutation carriers were < 45 years.

Other Cancer Types

Among 25 (12.95%) patients with other malignancies, P variants were most commonly observed in POLE (8.0%, n = 2), followed by NBN, BRCA2, RB1, VHL, and TSC2 (4.0% each, n = 1).

Variants of Uncertain Significance

Overall, 8.8% of women had variants (17/193) classified as VUS. VUS were most commonly observed in RAD50 (2.7% of breast cancer cases), followed by POLD1, CHEK2, BRCA1, BRCA2, and MSH6 (each in 1.4% of cases).

Discussion

This study presents one of the most comprehensive germline mutation profiles in Indian women with cancer, encompassing a diverse spectrum of malignancies and utilizing a 146-gene NGS panel. Including certain types of tumors such as breast, ovarian, and endometrial tumors for the study allowed for a targeted evaluation of clinically actionable germline variants most relevant to hereditary cancer syndromes in Indian women, as these cancers have a strong hereditary link with BRCA1/2 and MMR gene mutations. Our findings reveal that 55 (28.5%) female cancer patients harbor P/LP germline variants—a rate comparable to international studies reporting mutation prevalence between 20 and 30% in hereditary cancer cohorts.[13] [14] [15] The findings underscore the significant prevalence and diverse spectrum of P germline variants in this population, supporting the crucial role of hereditary predisposition in cancer susceptibility.

Prevalence and Spectrum of Germline Mutations

In females, previous studies have shown the onset of breast cancer at a younger age (∼45 years) as compared with the incidence of endometrial cancers (∼57 years), which generally presents later. Large population studies show a positive correlation with our results, where the mean age at diagnosis in breast cancer is generally earlier, often in the 40s to early 50s, than in endometrial cancer.[16] [17] [18] Findings of our study align with Indian studies, which have shown that P and LP variants were most commonly detected in female patients (n = 55; 28.5%), predominantly in breast and ovarian cancers.[19] [20] [21] In contrast, the male cohort exhibited a lower prevalence of known actionable findings (n = 6; 11.8%), notably, in colorectal, renal, and other cancers, but not in prostate cancer.

Overall, 28.5% of patients in our sample population harbored at least one P/LP germline mutation, with higher rates being present in cases of endometrial cancer (30%), followed by ovarian (28.6%) and breast (28%) cancers. The rates closely align with prior international studies reporting mutation prevalence between 11 and 23% in hereditary cancer cohorts, particularly in HBOC, and endometrial cancer (42.5%).[16] [17] [18] The results provide valuable insights into the distribution of germline mutations in both male and female cancer patients, with variations across different cancer types and genders.

BRCA1 and BRCA2 mutations collectively accounted for 17.6% of all cases, reinforcing their role as the most commonly altered high-penetrance genes in HBOCs in the Indian population.[22] [23] The prevalence of BRCA1 mutations (10.88%) exceeded BRCA2 (6.74%), consistent with patterns observed in multiple Asian populations.[24] [25] [26] While BRCA1/2 dominate HBOC risk globally (the United States, UK, China, Pakistan, and Brazil), other moderate-risk germline alterations in TP53, PALB2, CHEK2, ATM, RAD51C/D, and BRIP1 also contribute variably, reflecting population diversity.[27] [28] [29] [30]

Among breast cancer patients, BRCA1 mutations were most frequent (12.2%), particularly in triple-negative cases, which aligns with established genotype–phenotype correlations. The significant enrichment of P variants in early-onset disease (< 45 years) underscores the importance of genetic testing for young women with cancer, as recommended by current guidelines.[23] [31] [32]

Ovarian cancer patients exhibited a similarly high burden of germline mutations (28.6%), predominantly in BRCA1 (17.5%) and BRCA2 (9.5%). These rates are consistent with international reports, including those from ethnically diverse cohorts.[33] [34] The detection of TP53 and TSC2 mutations, though less frequent, highlights the broader mutational spectrum beyond BRCA genes in high-grade serous carcinomas, which may contribute to treatment resistance and prognosis.

Although Indian-specific data on germline predisposition in endometrial cancer remain limited, the well-established association between MMR genes and endometrial carcinogenesis in Western cohorts is likely relevant to the Indian context.[35] In our NCCN criteria–selected cohort, 30.0% of endometrial cancers harbored P/LP germline variants, predominantly in MMR genes—MLH1, MSH2, and MSH6—implicating Lynch syndrome as a key contributor. This yield exceeds rates reported in unselected endometrial cancer populations (approximately 2–6%) and likely reflects enrichment due to guideline-based testing.[36] Notably, one patient carried a P BRCA1 variant with retained MMR expression, highlighting a rare but possible non–MMR germline predisposition in endometrial cancer and underscoring the need for larger datasets to clarify risk estimates. IHC for MMR proteins was available in eight tumors with MMR protein loss; seven of eight (87.5%) showed loss concordant with the mutated gene, supporting the functional impact of these alterations and reinforcing the clinical utility of universal MMR screening in endometrial cancers in India.[37] Microsatellite instability testing was not uniformly applied and, accordingly, is not summarized at the cohort level to avoid conflating tumor and germline findings. Most carriers of P variants were diagnosed after the age of 45 years, aligning with the older age at presentation typically observed for endometrial cancers compared with breast cancers in this cohort.

Across all cancer types, VUS constituted a substantial proportion (8.8% of detected variants). The high VUS burden, especially in non-BRCA genes, highlights a critical challenge in the Indian population, where underrepresentation in global databases limits confident classification. This underscores the urgent need for population-specific variant databases and functional assays to aid in variant reclassification and genetic counseling.

Clinical Implications and Therapeutic Relevance

In this study, we identified mutations of BRCA1 (10.9%), BRCA2 (6.7%), and MMR genes (MLH1, MSH2, MSH6; cumulative 4.7%), along with additional variants in TP53, PTEN, PALB2, NBN, POLE, FANCA, TSC2, RB1, and VHL which are clinically actionable, as they influence therapy selection and genetic counseling. A total of 17 variants (8.8%) were classified as VUS, and they currently lack clinical treatment recommendations.

The identification of actionable germline mutations has profound implications for patient management and familial risk assessment. The first-degree relatives (parents, siblings, children) have a 50% chance of carrying the same P variant; thus, cascade testing of family members, timely surveillance, and application of risk-reducing surgeries can be beneficial.

For BRCA1/2 mutation carriers, prophylactic interventions such as risk-reducing salpingo-oophorectomy and enhanced surveillance can significantly decrease cancer-specific mortality. Furthermore, these patients may benefit from targeted therapies such as PARP inhibitors, which have demonstrated improved progression-free survival in BRCA-mutated ovarian and breast cancers.

Similarly, the detection of MMR gene mutations enables Lynch syndrome diagnosis, warranting colonoscopy surveillance and consideration of prophylactic hysterectomy for female carriers. MMR deficiency also predicts response to immune checkpoint inhibitors, offering therapeutic options for these patients.

Beyond established high-penetrance genes, our identification of mutations in moderate-risk genes such as PALB2, NBN, and POLE informs personalized surveillance strategies and contributes to the evolving understanding of cancer predisposition beyond BRCA1/2.

Challenges in Variant Interpretation

The high proportion of VUS (8.8% of detected variants) highlights a critical challenge in genetic testing within the Indian population. This underscores the urgent need for population-specific variant databases. Expanding Indian genomic repositories would facilitate more accurate variant classification by providing ethnically relevant allele frequencies. There is a need for high-throughput functional assays for VUS characterization to enhance clinical interpretation, along with collaborative efforts among Indian cancer centers to pool genetic data to accelerate VUS reclassification and improve genetic counseling.

These initiatives are particularly important given India's genetic diversity and the limited representation of South Asian populations in major genomic databases such as gnomAD and ClinVar.

Public Health Implications

Our findings have significant public health implications for cancer control in India. The high prevalence of actionable germline mutations supports the integration of genetic testing into routine cancer care, particularly for breast, ovarian, and endometrial malignancies. Implementation of cascade testing in families of mutation carriers represents a cost-effective strategy for identifying at-risk individuals and enabling targeted prevention.

Moreover, the distinctive pattern of hereditary mutations in Indian women can inform the development of population-specific genetic screening guidelines, which may differ from those optimized for Western populations. Such tailored approaches are essential given India's unique cancer epidemiology, characterized by a higher burden in females and distinctive risk factor profiles.

Limitations and Future Directions

Several limitations of this study warrant consideration. First, patient selection based on NCCN criteria may have enriched the cohort for hereditary cancers, potentially limiting generalizability to unselected cancer populations. Furthermore, our use of a target-enrichment panel, the GALEAS HereditaryPlus Kit, is designed around international guidelines and knowledge of current databases. While target enrichment strategies reduce sequencing cost, a key consideration for any study, we were unable to find novel mutated genes that may have been detected following a whole-genome approach. Second, the cross-sectional design precluded assessment of longitudinal outcomes, including therapeutic response and survival, which are critical end points for establishing clinical utility. Finally, India's genetic heterogeneity necessitates broader geographic sampling to fully characterize mutation patterns across the subcontinent, which cannot be adequately represented by analysis at a single tertiary care center. Therefore, to fully validate the findings of this study, a similar study should be repeated in a multicenter format. Microsatellite instability testing was not uniformly applied; future studies should standardize tumor testing across endometrial cases to enable integrated germline–somatic analyses.

Conclusion

This study has focused on hereditary cancer predisposition—unlike somatic, germline mutations are more important as they influence both clinical management and familial risk across generations. Our study demonstrates that more than one-quarter of Indian women with breast, ovarian, and endometrial cancers harbor clinically actionable germline mutations, primarily in BRCA1/2 and MMR genes. These findings underscore the importance of incorporating multigene panel testing into routine oncology practice in India and developing population-specific genomic resources to enhance variant interpretation.

The high prevalence of P variants, particularly in young patients and those with specific histological subtypes such as TNBC and high-grade serous ovarian cancer, highlights the potential for genetic risk stratification to guide precision prevention and treatment. Implementation of systematic cascade testing in families of mutation carriers represents a feasible approach to reducing cancer burden through early detection and intervention.

As India confronts its growing cancer epidemic, integrating hereditary cancer assessment into national cancer control programs will be essential for addressing the disproportionate burden of female malignancies. Our findings provide a foundation for such initiatives, emphasizing the unique genetic landscape of cancer predisposition in Indian women and the imperative for expanded access to genetic services across the subcontinent.

Conflict of Interest

None declared.

Acknowledgments

The authors gratefully acknowledge all patients who participated in this study and their families for their valuable contribution to advancing precision oncology in India.

Patient Consent

Written consent has been taken from the patient.

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Publication History

Article published online:
20 January 2026